The invention is directed to a method and devices for the in vitro production of arrangements of cell layers.
Various methods and devices are known for the in vitro production of arrangements of cell layers and culturing thereof. Cell culture inserts only allow cellular processes to be studied under static conditions. Accordingly, there is an enrichment of catabolic substances over the respective cell cultures so that their growth and vitality are reduced and limited in time. Since culturing is possible only under static conditions, no shear forces can be generated through liquids at the cells. However, these shear forces are necessary for a large number of cells so that they adopt physiologically correct properties such as occur, for example, in the human body.
Cells and tissues can be perfused in the Boyden chamber so that enrichment of catabolic substances is prevented, fresh nutritive media can be supplied to the cells or tissues, and the above-mentioned shear forces can be generated at the cells or tissues. However, this system has the grave disadvantage that the cells are efficiently perfused by nutrient medium from one side only (mostly apical). The cells cannot be perfused basolaterally. This also reduces and temporally limits cell growth and cell vitality. Further, the system is limited to culturing at most a two-dimensional cell area or tissue area.
Due to its design features, the arrangement described in US 2011/0091930 A1 allows the culturing of a maximum of only one cell layer. Further, as a result of the construction, no direct microscopy of the cells can be carried out under perfusion conditions in the well because supplying channel systems below the surface of the culture impede the optical observation axis of the microscope.
While the arrangement described in DE 10 2009 035 502 A1 allows cells and tissues to be observed under flow conditions, the wells cannot be perfused individually through separate liquid flows. Further, because of its design features, the system is not suitable for providing a plurality of culturing surfaces for cells in a well so that the interaction between different cell layers, which is important for a realistic picture of the functioning of organs in vitro, cannot be sufficiently taken into account.
The technical teaching from U.S. Pat. No. 8,357,528 B2 allows culturing of oppositely located cell layers on a porous formation such as a membrane, for example. The membrane separates two culture chambers from one another vertically. Based on the construction of the chamber system, it is not possible, for example, to observe the adhesion and/or transmigration of cells directly through a microscope. The system is limited to receiving two cell layers. Usually, at least three cell layers are required to model a multilayered organoid structure.
Possibilities described in US 2007/0037277 A1, US 2009/0023608 A1 and US 2010/0216244 A1 are also limited to two cell layers.